Meta Gene 7 (2016) 56–63

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Genetic diversity and molecular evolution of Naga King Chili inferredfrom internal transcribed spacer sequence of nuclear ribosomal DNA

Mechuselie Kehie a,⁎, Suman Kumaria b, Khumuckcham Sangeeta Devi b, Pramod Tandon b

a Post Graduate Department of Environmental Science, Patkai Christian College (Autonomous), Chumukedima, Seithekema, Dimapur 797103, Indiab Plant Biotechnology Laboratory, Centre for Advanced Studies in Botany, North-Eastern Hill University, Shillong 793022, India

⁎ Corresponding author.E-mail address: kehie10@gmail.com (M. Kehie).

http://dx.doi.org/10.1016/j.mgene.2015.11.0062214-5400/© 2015 The Authors. Published by Elsevier B.V

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 September 2015Revised 17 November 2015Accepted 30 November 2015Available online 2 December 2015

Sequences of the Internal Transcribed Spacer (ITS1-5.8S-ITS2) of nuclear ribosomal DNAswere explored to studythe genetic diversity andmolecular evolution of Naga KingChili. Our study indicated the occurrence of nucleotidepolymorphism and haplotypic diversity in the ITS regions. The present study demonstrated that the variability ofITS1with respect to nucleotide diversity and sequencepolymorphismexceeded that of ITS2. Sequence analysis of5.8S gene revealed amuch conserved region in all the accessions of Naga King Chili. However, strong phylogenet-ic information of this species is the distinct 13 bp deletion in the 5.8S gene which discriminated Naga King Chilifrom the rest of the Capsicum sp. Neutrality test results implied a neutral variation, and population seems to beevolving at drift–mutation equilibrium and free from directed selection pressure. Furthermore, mismatch analy-sis showedmultimodal curve indicating a demographic equilibrium. Phylogenetic relationships revealed by Me-dian Joining Network (MJN) analysis denoted a clear discrimination of Naga King Chili from its closest sisterspecies (Capsicum chinense and Capsicum frutescens). The absence of star-like network of haplotypes suggestedan ancient population expansion of this chili.

© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:Naga King ChiliPhylogeneticSequence analysisNucleotide diversityHaplotype

1. Introduction

Chili is one of themost important crops of theworld and has the dis-tinction of being the first plant to be cultivated in the new world. It hasbeen a part of the human diet since at least 7500 BC. Though tropicalSouth America is believed to be the original home of chili (Greenleaf,1986), chilies are now grown worldwide. The cultivated species of thegenus are Capsicum annuum, Capsicum chinense, Capsicum frutescens,Capsicum baccatum, Capsicum pubescens. Chili fruits are rich sources ofmetabolites such as carotenoids (provitamin A), vitamins (C and E), fla-vonoids and capsaicinoids that are beneficial for human health (Maga,1975; Ramchiary et al., 2014). Naga King Chili is considered as India'shottest chili and was formerly acknowledged as the world's hottestchili measuring 1,001,304 Scoville Heat Units (SHU) (Guinness BookofWorld Records, 2006; Kehie et al., 2012a). However, this chili was su-perseded by the Infinity chili in 2011, followed by the Naga Viper, theTrinidad Moruga Scorpion in 2012, and the Carolina Reaper in 2013(Hottest Chili, 2015). Naga King Chili which is known in local Angamidialect as ‘Kedi Chüsi’ which literally means the ‘King of Chilies’, is alsoknown by other local names such as Naga Mirchi, Bhut Jolokia, andUmorok (Kehie et al., 2013). Naga King Chili is native to the Northeast-ern states of India. Since time immemorial, people of Northeastern

. This is an open access article under

region, particularly the Naga people have close sodality with this chili.Nagas are kenned to have utilized this chili as remedial agents fortreatingmyriads of ailments. The Government of Nagaland has patentedthis chili and has registered as the proprietor with the Government ofIndia under Geographical Indication Registry (Kehie et al., 2014). Al-though earlier studies have treated this chili as C. frutescens, the taxo-nomic relationship of ‘Naga King Chili’ based on RAPD markers haveplaced ‘Naga King Chili’ in a taxonomic position between C. chinenseand C. frutescens with ‘Naga King Chili’ clustering more closely to theC. chinense group (Bosland and Baral, 2007). Internal TranscribedSpacers (ITS) sequence analysis reported by Purkayastha et al. (2012)showed distinct genetic differences between Bhut Jolokia and the spe-cies of C. frutescens and C. chinense. The occurrence of high cross pollina-tion and adaptation to micro-climatic conditions has led to theformation of variants and landraces within the species (Kehie et al.,2012b). However, study on its genetic diversity andmolecular evolutionremains unexplored as far as our knowledge is concerned. Molecularapproach using ITS of nuclear ribosomal DNA (nrDNA) has been widelyused for resolving phylogenetic relationships among closely relatedspecies of angiosperms. Eukaryotic nrDNA has two internal transcribedspacers ITS1 and ITS2. The two spacers and the 5.8S subunit are collec-tively known as the ITS region and it has become an important nuclearlocus for molecular systematic investigations of flowering plants. Theyhave frequent insertions/deletions which could be phylogenetically in-formative (Baldwin et al., 1995). The popularity of the ITS region can

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Table 1Sources of Capsicum ITS sequences and their geographical origin.

Species/cultivar GenBank accession no. Geographic origin Elevation (m) References

Source/location

Naga King Chili KP006659 Piphema, Nagaland, India 750 This studyNaga King Chili KP006658 Kohima, Nagaland, India 1444 This studyNaga King Chili KP006660 Ruzaphema, Nagaland, India 500 This studyNaga King Chili KP006657 Jalukie, Nagaland, India 347 This studyNaga King Chili KP006661 Shillong, Meghalaya, India 1525 This studyNaga King Chili KP006656 Imphal, Manipur, India 786 This study

GenBank sequencesBhut Jolokia HQ705983 Dibrugarh, Assam, India 108 Purkayastha et al. 2012Bhut Jolokia HQ705984 Jorhat, Assam, India 91 Purkayastha et al. 2012Bhut Jolokia HQ705985 Sonitpur, Assam, India 21 Purkayastha et al. 2012Bhut Jolokia HQ705986 Karbianglong, Assam, India 1600 Purkayastha et al. 2012Bhut Jolokia HQ705987 Ukhrul, Manipur, India 1662 Purkayastha et al. 2012Bhut Jolokia HQ705988 Kohima, Nagaland, India 1500 Purkayastha et al. 2012Capsicum frutescens HQ705989 Tezpur, Assam, India 48 Purkayastha et al. 2012Capsicum chinense HQ705990 Sonitpur, Assam, India 21 Purkayastha et al. 2012Capsicum eximium AY665841 Mexico, North America – Whitson and Manos, 2005Capsicum annum GU944973 Badajoz, Spain – Hernández et al. 2010Capsicum baccatum AF244708 Utah, USA – Bohs and Olmstead, 2001Capsicum pubescens AY875749 Madison, USA – Spooner et al. 2005Capsicum lycianthoides DQ314158 Madison, USA – Smith and Baum, 2006

= Not specified.

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be attributed to the relatively high rate of nucleotide substitution in thetranscribed spacers, permitting the systematic comparison of relativelyrecently diverged taxa (Ghada et al., 2013). The ITS region is influencedby concerted evolution which homogenizes the tandem copies withinindividuals, thus making ribosomal DNA amenable for phylogenetic in-ference (Hillis andDavis, 1988; Chiang and Schaal, 2000). However, var-iations within individuals have been reported primarily as a result ofslow concerted evolution (Harris and Crandall, 2000; Coté and Peculis,2001). In this study, we attempted to explore and investigate the genet-ic diversity and molecular evolutionary history of Naga King Chili usingnrDNA.

2. Methods

2.1. Taxon sampling and genomic DNA isolation

Samples of Naga King Chili were collected from different geograph-ical regions of Northeastern India viz., Nagaland,Manipur andMeghala-ya (Table 1). Total genomic DNA was extracted from frozen fruits ofNaga King Chili following the method described by Doyle and Doyle(1987)with someminormodifications. The isolated DNA concentrationwas estimated using a UV spectrophotometer (Perkin Elmer Lambda35) and its integrity was checked by agarose (1%) gel electrophoresis.

2.2. PCR targeting ITS region and sequencing

A polymerase chain reaction (PCR) was used to amplify the ITS re-gions. The ITS1, 5.8S, and ITS2 regions of Naga King Chili were amplifiedusing PCR primers ITS 4 and ITS 5 (White et al., 1990). The DNA

Table 2Length and G + C content of ribosomal DNA sequences of Naga King Chili.

Species/cultivar GenBank accession number ITS1

%GC Length

Naga King Chili KP006659 65.5 240Naga King Chili KP006658 62.5 240Naga King Chili KP006660 65.14 241Naga King Chili KP006657 65 240Naga King Chili KP006661 64.16 240Naga King Chili KP006656 65 240

amplification was performed in an Applied Biosystems Gene Amp®PCR System 2700, Rotkreuz, Switzerland. PCR reaction volume was25 μL, containing 5 μL of template DNA (50ng/ μL), 2 μL 10× PCR buffer(Tris with 15 mM MgCl2), 1.5 mM MgCl2, 5 μL primer at 10 pmol, 5 μLdNTPs at 2 mM, 0.2 μL Taq DNA polymerase at 3 U/μL, and 1.3 μLddH2O. The thermocycling protocol consisted of a single denaturationstep at 95 °C for 5 min, followed by 40 cycles of 1 min denaturation at94 °C, 1 min annealing at 57 °C, 2 min extension at 72 °C, with a final in-cubation step at 72 °C for 10 min. To confirm successful amplificationand size of the amplified fragment, 10 μL of the PCR product was runon 1% agarose gel in 1× TAE buffer, stained with ethidium bromide,and visualized under UV light. GeneRule 100 bp Plus DNA Ladder,ready-to-use, 100–3000 bp (Fermentas, In., Glenn Burnie, MD, USA).The PCR amplified product approximately 750 bp corresponding to18S partial, ITS1, 5.8S, ITS2 and 26S partial sequence were sequencedat theMacrogen Inc. (Seoul, Korea). Ampliconswere sequenced directlyin both sense and antisense directions. The amplification primers wereused as the sequencing primers.

2.3. Sequence alignments and phylogenetic analyses

All sequence information has been deposited in the GenBank data-base (accession no. KP006656, KP006657, KP006658, KP006659,KP006660, KP006661). Sequences of the whole ITS region of NagaKing Chili were used to determine ITS1, 5.8S and ITS2 boundaries by ho-mologous blast with published ITS sequences in the GenBank database.The ITS sequence alignmentswere performed using ClustalX version 2.1(Thompson et al., 1997). Alignments were subsequently adjusted man-ually using BioEdit version 7.2.5 (Hall, 1999). The length and GC content

5.8S ITS2 ITSEntire region

%GC Length %GC Length %GC Length

53.52 142 68.48 237 63.87 62053.52 142 68.64 236 62.78 61853.52 142 68.64 236 63.81 61952.81 142 68.64 236 63.59 61853.52 142 68.64 236 63.43 61853.52 142 69.06 236 63.19 618

Table 3Maximum composite likelihood estimate of the pattern of nucleotide substitution.

Original nucleotide

A T C G

(a)A – 0.09 0.21 42.89T 0.13 – 17.96 0.18C 0.13 7.34 – 0.18G 30.61 0.09 0.21 –

(b)A – 0.04 0.05 51.54T 0.05 – 0.05 0.05C 0.05 0.04 – 0.05G 48 0.04 0.05 –

(c)A – 0.04 0.08 62.5T 0.03 – 7.89 0.06C 0.03 3.92 – 0.06G 25.28 0.04 0.08 –

(d)A – 1.74 3.36 35.86T 1.86 – 14.21 2.93C 1.86 7.35 – 2.93G 22.81 1.74 3.36 –

(a) ITS1 spacer, (b) 5.8S gene and (c) ITS2 spacer, (d) ITS entire region of ribosomal DNA.Each entry shows the probability of substitution (r) from one base (row) to another base(column). For simplicity, the sum of r values is made equal to 100. Rates of different tran-sitional substitutions are shown in bold and those of transversionsal substitutions areshown in italics.

58 M. Kehie et al. / Meta Gene 7 (2016) 56–63

for each sequencewere estimated using the Endmemo software (http://www. endmemo.com/bio/gc.php) (Table 2). Pairwise sequence diver-gence in ITS1, 5.8S and ITS2 regions was calculated according to theMaximum Composite Likelihood (MCL) (Tamura et al., 2007) (Table 3).

The basic genetic parameters of variation within species were calcu-lated. For Naga King Chili data, we estimated nucleotide polymorphism(θw) (Watterson, 1975) and diversity (π) (Nei, 1978). Genetic diversitywas quantified by indices of haplotypes diversity (Hd) (Nei and Tajima,1983), pairwise estimates of nucleotide divergence (Pi) (Jukes andCantor, 1969), and the average of nucleotide differences (k). The transi-tion/transversion ratio ti/tv was estimated using the following formulaR = [A ∗ G ∗ k1 + T ∗ C ∗ k2] / [(A + G) ∗ (T + C)] with A, G, C, T asthe corresponding frequencies of four nucleotides (Tamura et al.,2007) (Table 4).

Table 4Nucleotide diversity, sequence polymorphism and neutrality test based on ribosomal DNA of N

ITS1 5.8S

N 6 6S 20 1h 4 2hd 0.867 0.333θw 0.03650 (SD = 0.01855) 0.00308 (SD = 0.00π 0.02861(SD = 0.01488) 0.00235 (SD = 0.001k 6.867 0Pi(JC) 0.02995 0.00236k (i) 0.333 0.333Pi(i) 0.00138 0Pi(s) 0.02861 0.00235Tajima's D −0.93302(P b 0.10) 0.93302 (P N 0.10)Fu and Li's D* −1.36967(P b 0.10) −0.95015 (P N 0.10)Fu and Li's F* −1.48775(P b 0.10) −0.96473 (P N 0.10)Fu's Fs 1.798 −0.003

Fig. 1. Aligned nrDNA ITS sequence (ITS1-5.8S-ITS2) of Capsicum sp. The polymorphic sites in adeletion of 10 bp, 5 bp and 13 bp are shown with arrow.

Selection neutrality was tested by both Tajima's D (Tajima, 1989)and Fu and Li's D* and F* methods (Fu and Li, 1993). Demographic pa-rameters were assessed using the distribution of pairwise sequence dif-ferences (mismatch distribution) of Rogers and Harpending (1992) andsite frequency spectra (distribution of the allelic frequency at a site) ofTajima (1989) using the program DnaSP software version 4.0 (Rozaset al., 2003). Median joining (MJ) network analysis was conductedusing software NETWORK 4.6.1.0 (Bandelt et al., 1999; available athttp://www.fluxus-engineering.com).

3. Results

3.1. ITS sequence analysis

ITS sequence alignment of Naga King Chili and other Capsicum sp. se-quences from the GenBank revealed sequence variability in Capsicumsp. It was observed that the variable of ITS1 exceeded that of ITS2. Se-quence alignment results of Naga King Chili showed two sites deletionviz., 10 bp and 5 bp (indicated by arrow) in the ITS1 sequence region(Fig. 1); these deletions were also observed in sister species viz.,C. chinense and C. frustescens, which were clustered more closely toNaga King Chili. Sequence analysis of 5.8S gene which is 142 bp lengthshowed a much conserved region in all the accessions of Naga KingChili. However, sequence comparison of 5.8S with other Capsicum sp. re-vealed a distinct phylogenetic signalwhich discriminatedNagaKing Chilifrom the rest of the Capsicum sp. This phylogenetic information is attrib-utable to its distinct 13 bp deletion (indicated by arrow) in the 5.8S gene.

3.2. Sequence diversity, length variation and GC content of ITS sequences

The ITS sequence (ITS1-5.8S-ITS2) obtained from Naga King Chilishowed variations in both length and composition (Table 2). The ITS1spacer was 240 bp in all accessions except accession KP006660, whichwas 241 bp, with an average of 240.16 bp. The length of ITS2 spacersin all the accession was found to be 237 bp except the accessionKP006659, whichwas 236 bpwith an average of 236.16 bp. The GC con-tent of ITS1 ranged from 52.5 to 55.5%, while in ITS2 the GC contentranged from 68.48 to 69.06%. The 5.8S rDNA sequence region had a con-served length of 142 bp, and its GC content varied from 52.81 to 53.52%andwas significantly lower than ITS1 and ITS2 in length andGC content.The length of the entire ITS region ranged from 618 to 620 bp with GCcontent ranging between 62.78 to 63.87%.

aga King Chili.

ITS2 ITS Entire region

6 65 264 60.867 1

308) 0.00928 (SD = 0.00568) 0.01843 (SD = 0.00920)52) 0.00876 (SD = 0.00260) 0.01499 (0.00573)

2.067 9.2670.00883 0.015250.333 0.6670.00140 0.001070.00876 0.014991.03194 (P N 0.10) −1.13197(P N 0.10)−0.21471 (P N 0.10) −1.17008(P N 0.10)−0.25135 (P N 0.10) −1.27884(P N 0.10)−0.439 −1.075

ll accessions of Naga King Chili in comparison with other species are shown with (⁎). Sites

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3.3. Nucleotide composition variation, mutational events and test forneutrality

The ITS sequence (ITS1-5.8S-ITS2) analysis showed that the nucleo-tide frequencies are 18.85% for adenine (A), 17.56% for thymine (T),33.95% for cytosine (C), and 29.64% for guanine (G). The transition/transversion rate ratios are k1= 12.233 (purines) and k2= 4.231 (py-rimidines). The overall transition/transversion bias is R = 3.746. Therewas a total of 618 positions in the final dataset.

The analysis in Table 3 indicated that in Naga King Chili, transitionswere more frequent than transversions at the intergenic spacer in ITS.The results showed that G/A and A/G transition were more frequentthan C/T and T/C. The composition variation of nucleotide sequencealignment of ITSwithin amatrix of 621 characters showed the existenceof 592 conserved sites, 26 variable sites containing 3 informative sitesand 23 singleton sites. The sequence variation in the intergenic spacerITS of the ribosomal DNA was detected with 6 haplotypes observed for6 accessions of Naga King Chili. The haplotype diversity (Hd) wasfound to be 1 whereas nucleotide diversity (π) was 0.01499. Further-more, the average difference between pairs of nucleotide (k) was calcu-lated (9.267) which showed polymorphism (Table 4).

Neutrality test of Tajima (Tajima, 1989) and Fu and Li (Fu and Li,1993) statistics were employed to assess whether the sequencing datashows evidence of deviation from neutrality. We obtained negativevalues whichwere not significant, which is an indication of neutral var-iation, and population seems to be evolving at drift–mutation equilibri-um with no evidence of selection.

Further examination to assess deviation from neutrality, Fu statistics(Fu, 1997) was employed to detect neutrality. Sequence analysisshowed (Table 4; Fig. 2) positive and negative valueswhichwere statis-tically not significant for this parameter. Fu's Fs = 1.798 for ITS 1, Fu's

Fig. 2. Spectrum of frequencies of sites at the ribosomal DNA sequences in Naga King Chili. (a)spacer of ribosomal DNA (ITS2) and (d) intergenic spacer of ribosomal DNA (ITS). The solid lineThe index value of Fu is given.

Fs = −0.003 for 5.8S gene, Fu's Fs = −0.439 for ITS2 and Fu'sFs = −1.075 for ITS entire region. Fu and Li and Fu statistics indicatedno evidence for changes in population size or any particular pattern ofselection in the regions examined in Naga King Chili. Furthermore, oursuggestions were confirmed by the values of raggedness index “r”higher than 0.05 showing values of 0.1067 for the intergenic spacer ofribosomal DNA (ITS) (Fig. 3).

3.4. Mismatch distribution

Analysis of mismatch distribution in the ITS sequences of rDNA con-firmed a neutral variation. A frequency graph between alleles showingmultimodal aspect of the curve of mismatch indicated demographicequilibrium and not a recent demographic expansion (Fig. 3).

3.5. Haplotype network analysis

The haplotype network was reconstructed based on the whole ITSsequence data of Naga King Chili combining with ITS sequences of Cap-sicum sp. downloaded from GenBank. Median joining networks weredrawn for the haplotypes identified in 19 ITS sequences of Capsicumsp. The network illustrated the relationship between 18 haplotypes(Fig. 4). Evolutionary relationships among haplotypes were inferredusingMJN approach,which showed a clear separation between lineagesof Naga King Chili (H1, H2, H3, H4, H6, H7, H8, H9, H11, H12, H13) andthe rest of the other haplotypes of Capsicum sp. (H5, H10, H14, H15,H16, H17, H18). Haplotype network showed that majority of the NagaKingChili accessions seem to emerge fromH3,which is also representedby two accessions (KP006657, KP006660). It may also be inferred fromthe network analysis that haplotype represented by H13 (KP006656) isthe founder haplotype for H6 (KP006659), H7 (HQ705983), H9

Intergenic spacer of ribosomal DNA (ITS1), (b) 5.8S gene of ribosomal DNA, (c) intergenics in the spectrum indicate the distributions under neutrality and balance (mutation–drift).

Fig. 3. Graphs depicting the results of the mismatch distribution analysis for the sequences of rDNA based on the differences between pairs of sequences. (a) Intergenic spacer of rDNA(ITS1), (b) 5.8S gene of rDNA, (c) intergenic spacer of rDNA (ITS2) and (d) intergenic spacer of rDNA (ITS). The parameter for raggdness (r) is given. The observed frequencies were rep-resented by red dotted line. The solid green line corresponds to the frequency expected (Exp) under the hypothesis of population expansionmodel. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

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(KP006658), andH4 (KP006661) as basic brancheswere seen to be con-nected to this haplotype. Network analysis indicated that haplotype H5(C. chinense) and closely related haplotype 10 (C. frutescens) are likely tobe the ancestral haplotype of Naga King Chili accessions as they weremore closely clustered around these haplotypes.

4. Discussions

The ITS sequence region is one of the most popular loci used in mo-lecular phylogenetic studies (Alvarez andWendel, 2003). It is known toevolve relatively quickly and is used for determining inter-specific(Jorgenson and Cluster, 1988) and intra-specific relationships (Bauraet al., 1992). Our results showed that the variability of ITS1 with respectto nucleotide diversity and sequence polymorphism exceeded that ofITS2. Two sites deletion (10 bp and 5 bp) in the ITS1 sequence regionof Naga King Chili were also observed in sister species viz., C. chinense,C. frustescens and C. pubescens, which were clustered more closely toNaga King Chili.Within the ITS region, the ITS1 sublocus evolved slightlymore rapidly with a more variable length than the ITS2 sublocus (Hillisand Dixon, 1991; Hershkovitz and Lewis, 1996). Much attention hasbeen focused on ITS1 as the more variable sublocus of the two andthereby, presumably, the better species marker (Chen et al., 2001;Narutaki et al., 2002; Hinrikson et al., 2005). Sequence analysis of 5.8Sgene revealed a much conserved region in all the accessions of NagaKing Chili. As known, the 5.8S rRNA region is highly evolutionary con-served in interspecific and intraspecific levels (Hřibová et al., 2011).However, it is important to note that the 13 bp deletion in the 5.8Sgenewhich discriminated Naga King Chili from the rest of the Capsicumsp. provided strong phylogenetic information of this species. Our find-ings are in agreement with the earlier report of Purkayastha et al.,(2012), thus corroborated with more sequence sources of ITS regionand denoted a remarkable genetic difference of Naga King Chili withother Capsicum sp.

In our study to identify sequenceswhichdonot fit the neutral theorymodel at equilibrium between mutation and genetic drift, we usedTajima (Tajima, 1989) and Fu and Li tests (Fu and Li, 1993). Our resultsshowed negative values which were not significant, an indication ofneutral variation and population evolving at drift–mutation equilibriumwith no evidence of selection. The study was further supported by theassessment of Fu statistics (Fu, 1997). In addition, mismatch analysisof the frequency graph between alleles showing multimodal aspect ofthe curve indicated a demographic equilibrium and not a recent demo-graphic expansion. Phylogenetic methods may not lead to the desiredresolution at the intraspecific level due to lower genetic diversity andnon-hierarchical nature of intraspecific data sets (Posada and Crandall,2001) and complementary network approaches might be valuablealternatives to study phylogenetic structures and haplogroups at thepopulation level. Simulations (Woolley et al., 2008) have shown thatMJN (Bandelt et al., 1999) outperforms minimum spanning networks(MSN) because the former is able to infer ancestral haplotypes(Woolley et al., 2008). In the present study, haplotype network analysisshowed connections between haplotypes and summaries of all theshortest trees possible. Haplotypes of Naga King Chili accessions wereclustered separately suggesting that they are a descendant populationderived from a common ancestor. Majority of the Naga King Chili acces-sions seem to emerge from H3. Haplotype H5, H10 clustering closest tohaplotypes of Naga King Chili are likely to be the ancestral haplotype.The absence of star-like cluster of nodes in the network indicated anon-demographic expansion and further supported a demographicequilibrium.

5. Conclusion

The results of this study contributed to the knowledge of the existinggenetic status of Capsicum sp. Based on the sequence analysis of thenrDNA ITS region, the phylogenetic relationship of Naga King Chilishowed a clear grouping from C. chinense and C. frutescens. This work

Fig. 4. Median-joining network of the haplotypes inferred from ITS sequences. Nodes are proportional to haplotypes frequencies and branches length is proportional to the number ofmutations. The red dots represent theoretical median vectors introduced by the network software. H1: HQ705985, H2: HQ705988, H3: KP006657; KP006660, H4: KP006661, H6:KP006659, H7: HQ705983, H8: HQ705987, H9: KP006658, H11: HQ705986, H12: HQ705984, H13: KP006656, H5: C. chinense, H10: C. frutescens, H14: C. eximium, H15: C. annum, H16:C. baccatum, H17: C. pubescens, H18: C. lycianthoides. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

62 M. Kehie et al. / Meta Gene 7 (2016) 56–63

not only providedmore sequence sources of ITS region of Capsicum spe-cies but the basis for clearer discrimination of Naga KingChili fromotherrelated chili species. It appeared that Naga King Chili population isevolving at drift–mutation equilibrium and free from directed selectionpressure. Furthermore, our findings revealed an ancient evolutionaryhistory of this species.

Conflicts of interest

The authors declare no conflict of interest.

Authors' contributions

MKconceived anddesigned research.MK andKSD conducted exper-iments.MK, SK, PT contributed reagents or analytical tools.MK analyzeddata. MK. SK, PT wrote the manuscript. All authors read and approvedthe manuscript.

Acknowledgements

We gratefully acknowledge the Department of Science and Technol-ogy, Science and Engineering Research Board, Government of India, forfinancial assistance to Mechuselie Kehie, under the Scheme for Youngscientists, a prestigious start up research grant (SB/YS/LS-147/2013 dt.05-12-2013).We would like to thank Dr. Jeremy Dkhar for his untiringhelp and guidance during data analysis. We also wish to thank Ms. J.Sureni Yanthan for extending help during the study.

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